1. Field of the Invention
The invention pertains to the field of immunoassays. More particularly, the invention pertains to methods and devices for increasing specificity and/or accuracy of lateral flow immunoassays.
2. Description of Related Art
Lateral flow immunoassays are a subset of antibody-based immunoassays combining various reagents and process steps in one assay strip, thus providing a sensitive and rapid means for the detection of target molecules. Lateral flow immunoassays are available for a wide area of target analytes and can be designed for sandwich or competitive test principles. Generally high molecular weight analytes with several epitopes are analyzed in a sandwich format whereas small molecules representing only one epitope are detected by means of a competitive assay. The first tests were made for human chorionic gonadotropin (hCG). Today there are commercially available tests for monitoring ovulation, detecting infectious disease organisms, analyzing drugs of abuse and measuring other analytes important to human physiology. Products have also been introduced for veterinary testing, environmental testing and product monitoring.
U.S. Pat. No. 5,714,341 discloses a lateral flow immunoassay for HIV specific antibodies in saliva samples. The saliva sample is diluted in a sample buffer and the lateral flow immunoassay is dipped into the diluted saliva sample. The disclosure of this document is herein incorporated by reference.
German Patent DE 196 22 503 discloses methods for detecting illegal narcotics on a surface using lateral flow immunoassays. The disclosure of this document is herein incorporated by reference.
The growing use of antibody based immunoassays in recent years has required increased effort and investigation on minimizing interferences found in many samples. A typical problem is the presence of interfering substances, e.g. antibodies, in whole blood, serum and other human fluid samples. These interfering antibodies can be divided into auto-antibodies or rheumatoid factors (RF), heterophilic antibodies and human anti-mouse antibodies (HAMA).
Auto-antibodies or rheumatoid factors (RF) show anti-IgG activity and are predominantly composed of the IgM class. Most often they recognize the Fc region of the antigen bound IgG antibodies. Rheumatoid factor antibodies may also be of the IgG and IgA classes and have been observed reacting with antibodies of the IgM class. To further complicate this group of interfering antibodies, rheumatoid factors from one species may react with immunoglobulins of another species.
Heterophilic antibodies are one of many sources of interference in immunoassays. This often-misapplied term was historically used to refer to certain populations of antibodies in patient sera, which caused the aggregation of sheep red blood cells, and observed to be associated with Epstein-Barr virus (EBV) infections. In immunoassay development labs today, the term heterophile is frequently used to describe an interfering antibody (or other binding molecule) which has an unknown origin. These relatively common low affinity antibodies occur in approximately 1-5% of the healthy human population and effectively compete with the analyte of interest, which may produce abnormally high or false positive immunoassay results.
Human anti-mouse antibodies (HAMA) are high affinity human anti-animal antibodies, which are directed against specific animal immunoglobulins. Human anti-mouse antibodies have been reported to give false positive results in sandwich immunoassays that utilize mouse monoclonal IgG. HAMA reactivity has been detected in approximately 9% of the normal human population. In this segment of the population, the patient sample contains an antibody to mouse immunoglobulin due to a previous exposure to mouse antibodies. This can occur through diet or through exposure, or may be a direct result of monoclonal antibody therapy—a presently uncommon, but growing subset of the patient population. Actually, not all HAMAs are human anti-mouse antibodies. Many are other animals, such as, for example anti-rabbit antibodies or anti-dog antibodies. Since immunoglobulins are highly conserved across species, it is not uncommon to see a patient with an antibody titer to immunoglobulins exhibiting cross-reactivity to mouse IgG.
All these interfering antibodies are capable of simulating an analyte of interest when body fluids are tested in an immunoassay. This interference can result in false positives, false negatives and all graduations in between these two extremes. There is a need in the art for methods and devices that accurately detect analytes in body fluids in the presence of interfering substances.